Use Of Amplified Fragment Length Polymorphism Research Articles

Amplified fragment length polymorphism: an adept technique for genome mapping, genetic differentiation, and intraspecific variation in protozoan parasites

With the advent of polymerase chain reaction (PCR), genetic markers are now accessible for all organisms, including parasites. Amplified fragment length polymorphism (AFLP) is a PCR-based marker for the rapid screening of genetic diversity and intraspecific variation. It is a potent fingerprinting technique for genomic DNAs of any origin or complexity and rapidly generates a number of highly replicable markers that allow high-resolution genotyping. AFLPs are convenient and reliable in comparison to other markers like random amplified polymorphic DNA, restriction fragment length polymorphism, and simple sequence repeat in terms of time and cost efficiency, reproducibility, and resolution as it does not require template DNA sequencing. In addition, AFLP essentially probes the entire genome at random, without prior sequence knowledge. So, AFLP markers have emerged as an advance type of genetic marker with broad application in genomic mapping, population genetics, and DNA fingerprinting and are ideally suited as screening tool for molecular markers linked with biological and clinical traits. This review describes the AFLP procedure and its applications and overview in the fingerprinting of a genome, which has been currently used in parasite genome research. We outline the AFLP procedure adapted for Leishmania genome study and discuss the benefits of AFLPs for assessing genetic variation and genome mapping over other existing molecular techniques. We highlight the possible use of AFLPs as genetic markers with its broad application in parasitological research because it allows random screening of the entire genome for linkage with genetic and clinical properties of the parasite. In this review, we have taken a pragmatic approach on the study of AFLP for genome mapping and polymorphism in protozoan parasites and conclude that AFLP is a very useful tool.

Molecular Characterization of Acinetobacter Isolates Collected in Intensive Care Units of Six Hospitals in Florence, Italy, during a 3-Year Surveillance Program: a Population Structure Analysis

The strain diversity and the population structure of nosocomial Acinetobacter isolated from patients admitted to different hospitals in Florence, Italy, during a 3-year surveillance program, were investigated by amplified fragment length polymorphism (AFLP). The majority of isolates (84.5%) were identified as A. baumannii, confirming this species as the most common hospital Acinetobacter. Three very distinct A. baumannii clonal groups (A1, A2, and A3) were defined. The A1 isolates appeared to be genetically related to the well-characterized European EU II clone. A2 was responsible for three outbreaks which occurred in two intensive care units. Space/time population dynamic analysis showed that A1 and A2 were successful nosocomial clones. Most of the A. baumannnii isolates were imipenem resistant. The genetic determinants of carbapenem resistance were investigated by multiplex PCR, showing that resistance, independently of hospital origin, period of isolation, or clonal group, was associated with the presence of a bla (OXA-58-like) gene and with ISAba2 and ISAba3 elements flanking this gene. bla (OXA-58) appeared to be horizontally transferred. This study showed that the high discriminatory power of AFLP is useful for identification and typing of nosocomial Acinetobacter isolates. Moreover the use of AFLP in a real-time surveillance program allowed us the recognition of clinically relevant and widespread clones and their monitoring in hospital settings. The correlation between clone diffusion, imipenem resistance, and the presence of the bla(OXA-58-like) gene is discussed.

Combined use of Amplified Fragment Length Polymorphism and IS6110 -RFLP in fingerprinting clinical isolates of Mycobacterium tuberculosis from Kerala, South India

BackgroundDNA fingerprinting by IS6110-RFLP has shown a high incidence of Mycobacterium tuberculosis isolates having no and low copies of the insertion sequence in Kerala, South India. Amplified Fragment Length Polymorphism (AFLP) would scan the entire genome rather than a few repetitive elements, we thought that this technique would help us in differentiating the large reservoir of isolates from an endemic region. Here we evaluate the ability of Amplified Fragment Length Polymorphism (AFLP) to type clinical isolates.MethodsFifty clinical isolates of M. tuberculosis were analysed by conventional radioactive AFLP and IS6110- RFLP. M. bovis, M. bovis BCG and two non tuberculous mycobacteria were also analysed to see species specific differences generated by AFLP. Cluster analysis was performed using the AFLP profile that showed the maximum polymorphism within M. tuberculosis and this was compared to the number of copies of IS6110 insertions.ResultsFor AFLP, out of ten primer pairs tested, the EO/MC pair generated maximum polymorphism among the clinical isolates of M. tuberculosis. The similarity between the isolates ranged between 88 and 99.5%. Majority (nearly 85%) of the 'low copy' IS6110 isolates clustered together, while the rest clustered irrespective of the copy numbers. AFLP could show rare differences between isolates of M. tuberculosis, M. bovis and M. bovis BCG. The AFLP profiles for non-tuberculous mycobacteria were highly different from those of M. tuberculosis.ConclusionPolymorphism generated by AFLP within the M. tuberculosis species is limited and hence AFLP alone seems to have limited use in fingerprinting the isolates in Kerala. The combined use of AFLP and IS6110-RFLP showed relatively better differentiation of 'high copy' IS6110 isolates, but failed to differentiate the 'low copy' isolates. However, the technique may be efficient in inter-species differentiation, and hence potentially useful in identifying and developing species- specific markers.

Use of AFLP for differentiation of Metschnikowia pulcherrima strains for postharvest disease biological control

Metschnikowia pulcherrima occurs naturally on fruits, buds and floral parts of apple trees. Some strains are effective as biocontrol agents against postharvest decay of apples and other fruits. The usefulness of the amplified fragment length polymorphism (AFLP) technique was evaluated for the genetic analysis of 26 strains of M. pulcherrima, isolated from different sources in different geographical regions. With six AFLP primer pairs, 729 polymorphic bands were scored. The technique showed a high discriminatory power. Genetic relationships between strains were also estimated using AFLP. All the isolates from the carposphere of apple, previously tested as biocontrol agents, were grouped in a single cluster with a high bootstrap value (97), indicating robustness and reproducibility. AFLP patterns could clearly distinguish the different strains and research is in progress to use some putative specific bands for single tag sequence (STS) conversion to develop isolate-specific markers.

Genetic fingerprinting of Flavobacterium columnare isolates from cultured fish.

To evaluate the intraspecific diversity of the fish pathogen Flavobacterium columnare. Genetic variability among Fl. columnare isolates was characterized using restriction fragment length polymorphism analysis of the 16S rDNA gene, intergenic spacer region (ISR) sequencing, and amplified fragment length polymorphism (AFLP) fingerprinting. Thirty Fl. columnare cultures isolated from different fish species and geographical origins as well as reference strains were included in the study. Fifteen isolates belonged to genomovar I while eleven were ascribed to genomovar II. Analysis of the ISR sequence confirmed the genetic differences between both genomovars but revealed a higher diversity among genomovar I isolates. The maximum resolution was provided by AFLP fingerprinting, as up to 22 AFLP profiles could be defined within the species. We confirmed the division of Fl. columnare isolates from cultured fish into different genogroups. We showed that both genomovars I and II are present in channel catfish from the US. We described a unique genetic group represented by four Fl. columnare isolates from tilapia in Brazil which appears to be related to both genomovars. We were able to further subdivide the species by analysing the ISR. Finally, the use of AFLP allowed us to fingerprint the species at clone level without losing the higher genetic hierarchy of genomovar division. This paper reports on an extensive assessment of the use of molecular tools for the study of the epidemiology of the fish pathogen Fl. columnare.

Genetic diversity among selected Argentinean garlic clones (Allium sativum L.) using AFLP (Amplified Fragment Length Polymorphism)

Genetic diversity of eight selected Argentinean garlic clones (Allium sativum L.) were investigated at the DNA level with the amplified fragment length polymorphism DNA (AFLP) procedure. A total of 405 unambiguous bands were identified by six primer combinations of EcoR I +3 and Mse I +3, of those, 398 showed a clear polymorphism, representing 98% of the total bands. A presence/absence matrix was constructed with the polymorphic bands, and a dendrogram was obtained from it with the UPGMA method. The accessions showed different levels of similarity ranging between 0.24 and 0.97, using the coefficient of Jaccard. The dendrogram showed six arbitrary groups. Accessions typically considered as different clones show similarities between 0.97 and 0.495. The garlic clones were clustered according to the physiological group and bulb color. We could detect an association between AFLP and the geographical origin of the clones. The potential use of AFLP could allow not only the differentiation among species, but also between botanical varieties and well-defined ecotype groups. This is the first report of the use of AFLP to characterize Argentinean garlic clones.

The use of AFLP to find an informative SNP: genetic differences across a migratory divide in willow warblers.

We used the amplified fragment length polymorphism (AFLP) method to obtain genetic markers distinguishing two subspecies of willow warblers Phylloscopus trochilus that have different migratory behaviours but are not differentiated in mitochondrial DNA or at several microsatellite loci. With the inverse-polymerase chain reaction (PCR) approach we converted a dominant AFLP-marker to a codominant single nucleotide polymorphism (SNP). Across Scandinavia we typed 621 birds at the SNP locus AFLP-WW1 and we found a sigmoid change in allele frequencies centred around 62 degrees latitude. North of the latitudinal cline was a west-east cline. Both clines are narrower than one would expect from dispersal distances in willow warblers, which suggests that these are maintained by selection. The latitudinal cline at the locus AFLP-WW1 is paralleled by changes in several other traits, all of which might be maintained by a single selective force. The most plausible selection factor that we have identified is selection against hybrids because of inferior migratory behaviour. The selective force maintaining the east-west cline is less obvious. We discuss alternatives to the selection scenario, involving colonization history and asymmetric gene flow.

The use of AFLP to relate cheese-contaminating Penicillium strains to specific points in the production plants

Amplified fragment length polymorphism (AFLP) analysis was performed on isolates of Penicillium commune and Penicillium palitans originating from cheese and indoor environment in four cheese factories. The AFLP method was found to be a useful tool for identification of P. commune and P. palitans on, as well as below, species level. However, AFLP in combination with M13 fingerprinting described in a previous paper provided better resolution at the intraspecific level than either of the methods alone. Specific P. commune and P. palitans strains were found in the same factories over a period of more than a year and showed that the cheese factories have contaminating strains that are well established. The majority of the P. commune and P. palitans strains were found only within a single factory, but several were found in different cheese factories. The combined fingerprinting data could relate strains isolated from cheese to specific points in the production plants. Several of cheese-contaminating Penicillium strains could be related to air in the wrapping room, which must be considered to be a critical point for contamination of cheese.

AFLP fingerprinting for assessing intraspecific variation and genome mapping in mites.

Molecular genetic techniques have come a long way in the last decade. With the advent of PCR, genetic markers are now accessible for all organisms, including mites. However, there is usually a trade-off between the accuracy of the molecular technique or genetic marker and expediency. In mites, many molecular techniques are not applicable due to their small size. Here we describe a relatively new molecular fingerprinting technique, amplified fragment length polymorphism (AFLP), which is currently used widely in plant genomic research. We outline the AFLP procedure adapted for mites, show results using this technique from our own research and discuss the benefits and limitations of AFLPs for assessing genetic variation and for genome mapping. It is our intention to highlight the possible use of AFLPs as genetic markers with a broad application in acarological research.

Amplified Fragment Length Polymorphism (AFLP) Provides Molecular Markers for the Identification of Caladium bicolor Cultivars

Caladiums are popular ornamental plants that have not been well studied at the molecular level. Identification of species within the genus Caladium (Araceae) has been based primarily on morphology. However, the lack of comprehensive references makes identification of Caladium cultivars extremely difficult. Amplified fragment length polymorphism (AFLP) analysis using 17 primer combinations was carried out on two species of Caladium (C. bicolor and C. schomburgkii), including six cultivars of C. bicolor. Results showed that AFLP can be used to distinguish these two species by their unique and different banding patterns. Unweighted Pair Group Method using Arithmetic Averages (UPGMA) permitted cluster analysis of data from 17 selected primer combinations on six cultivars of C. bicolor and one cultivar ofC. schomburgkii . It showed that closely related species can clearly be differentiated and that genetic difference between cultivars can also be established. Unique AFLP molecular markers were detected for all the C. bicolor cultivars used. The use of AFLP has potential for precisely characterizing and identifying particular caladium cultivars as well as for the registration of new cultivars. It will also be useful in future breeding programmes and systematics studies.

AFLP-derived, Codominant Markers for Locus-specific Applications

The amplified fragment length polymorphism (AFLP) is a powerful marker, allowing rapid and simultaneous evaluation of multiple potentially polymorphic sites. Although well-adapted to linkage mapping and diversity assessment, AFLPs are primarily dominant in nature. Dominance, relatively high cost, and technological difficulty limit use of AFLPs for marker-aided selection and other locus-specific applications. In carrot the Y2 locus conditions carotene accumulation in the root xylem. We identified AFLP fragments linked to the dominant Y2 allele and pursued conversion of those fragments to codominant, PCR-based forms useful for locus-specific applications. The short length of AFLPs (≈60 to 500 bp) precludes development of longer, more specific primers as in SCAR development. Instead, using sequence information from cloned AFLP fragments for primer design, regions outside of the original fragment were amplified by inverse PCR or ligation-mediated PCR, cloned, and sequenced. Differences in sequences associated with Y2 vs. y2 allowed development of simple PCR assays differentiating those alleles. PCR primers flanking an insertion associated with the recessive allele amplified differently sized products for the two Y2 alleles in one assay. This assay is rapid, technologically simple (requiring no radioactivity and little advanced training or equipment), reliable, inexpensive, and codominant. Our PCR assay has a variety of large scale, locus-specific applications including genotyping diverse carrot cultivars and wild and feral populations. Efforts are underway to improve upon conversion technology and to more extensively test the techniques we have developed.